Rapid and highly specific detection of site-specific 5-hydroxymethylcytosine based on peroxotungstate oxidation and mismatch ligation-based LAMP.

We have developed a rapid and specific method for site-specific 5-hydroxymethylcytosine (5hmC) quantification at single-base resolution. This bisulfite-free method integrates the peroxotungstate oxidization with the mismatched probe-assisted ligation to guarantee the specificity. Moreover, the high-efficiency LAMP also makes the proposed method suitable for the detection of low-content samples.

Amyloid-beta Targeted Therapeutic Approaches for Alzheimer's Disease: Long Road Ahead.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory impairment and cognitive decline. The obvious pathological features of AD are still amyloid plaques and neurofibrillary tangles. Development of disease-modifying treatments for AD has been challenging, with almost all drugs aborted. The amyloid cascade concept has been questioned due to the failures of various amyloid-targeting prospects. Despite this, targeting amyloid-ß (Aß) active immunotherapy provided some positive results to support this hypothesis and clinical trials of these candidates are ongoing. In this review, we describe the latest advance in therapeutic strategies based on amyloidogenic processing and evaluate the pros and cons of each treatment strategy. We also highlight the current status of the hottest immunotherapy and discuss the future development direction.

Identification of novel Aß-LilrB2 inhibitors as potential therapeutic agents for Alzheimer's disease.

LilrB2 is an Aß receptor with high affinity, which not only contributes to memory deficits but also mediates the loss of synaptic plasticity. Thus, Aß-LilrB2 interaction inhibitors (ALIs) might be a potential therapeutic strategy for Alzheimer's disease. In this study, an ELISA-based interaction assay was established as a novel approach to identify ALIs and was used to screen 110 compounds from a compound library. Among the 110 compounds, four compounds presented IC50 values lower than the positive control flusipirilene. The two phenyl-1,3,5-triazine derivatives (compound 103 and 104) displayed inhibitory activities with the IC50 of 0.23 µM and 0.05 µM respectively. The neuroprotection activities of the hit compounds were evaluated in SH-SY5Y cell line. Compound 104 presented good safety and neuroprotective effects against Aß. Further study of its effect on the downstream pathway of Aß indicated that compound 104 was able to reverse the Aß induced cofilin dephosphorylation, tau hyperphosphorylation and neurite outgrowth inhibition. The docking study showed that fluspirilene and compound 104 were favorably positioned into the Ben 3 and 4 binding pockets via their aromatic ring, which was similar to that reported for Aß. Based on these facts, compound 104 can be identified as a potential ALI which might be of therapeutic importance for AD treatment.

Therapeutic Strategies Targeting Amyloid-ß Receptors and Transporters in Alzheimer's Disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disease that has been recognized as one of the most intractable medical problems with heavy social and economic costs. Amyloid-ß (Aß) has been identified as a major factor that participates in AD progression through its neurotoxic effects. The major mechanism of Aß-induced neurotoxicity is by interacting with membrane receptors and subsequent triggering of aberrant cellular signaling. Besides, Aß transporters also plays an important role by affecting Aß homeostasis. Thus, these Aß receptors and transporters are potential targets for the development of AD therapies. Here, we summarize the reported therapeutic strategies targeting Aß receptors and transporters to provide a molecular basis for future rational design of anti-AD agents.

(m)RVD-hemopressin (α) and (m)VD-hemopressin (α) improve the memory-impairing effect of scopolamine in novel object and object location recognition tasks in mice.

Dysfunction of cholinergic system plays an important role in disease associated with cognitive blockage, such as Alzheimer's disease (AD). Central administration of scopolamine, an antagonist of acetylcholine receptor, could induce memory impairment in mice. Endocannabinoid system was also implicated in AD, as two peptides agonists of cannabinoid 1 receptor (CB1R), (m)RVD-hemopressin (α) (RVD) and (m)VD-hemopressin (α) (VD) have been reported to inhibit the AD-relating impairment in animal and cell models. More than one-third of the cholinergic cells expressed CB1R, so we speculated that RVD and VD might have ability to inhibit the memory-impairing effect of scopolamine. Our results showed RVD and VD ameliorated the memory toxicity of scopolamine, and the effects of the two peptides could be blocked by CB1R antagonists hemopressin (Hp) and AM251 in novel object and object location recognition tasks in mice. This study suggested that RVD and VD might be potential compounds for the treatment of the disease associated with impairment of cholinergic system.

AMPK alleviates oxidative stress­induced premature senescence via inhibition of NF-κB/STAT3 axis-mediated positive feedback loop.

Stress-induced premature senescence (SIPS) is characterized by the secretion of a variety of inflammatory cytokines, chemokines, and proteases, which are defined collectively as the senescence-associated secretory phenotype (SASP). AMP-activated protein kinase (AMPK) activation contributes to SIPS prevention, and the impact of AMPK on SASP may be included, but the mechanisms governing this phenomenon have not elucidated. In this study, we showed that SIPS is accompanied by a dynamic fluctuation of NF-κB activation, which induces SASP production, whilst reinforcing and amplifying local STAT3 signalling and subsequently enhancing downstream senescence. NF-κB and STAT3 inhibitors attenuate oxidative stress-induced senescence in a time-dependent manner. Conditioned medium (CM) from senescent cells rich in SASP factors can induce growth arrest and promote senescence in healthy cells; accordingly, a STAT3 inhibitor blunts the SASP-induced senescence, indicating a positive feedback mechanism via the NF-κB/STAT3 pathway that sustains SASP production and promotes senescence. In addition, we confirmed that AMPK negatively regulates SASP production and senescence development associated with NF-κB/STAT3 inhibition. In summary, our results suggest that AMPK prevents oxidative stress-induced senescence development via inhibiting the NF-κB/SASP/STAT3 signalling mediated positive feedback loop.

Effect of (m)VD-hemopressin against Aß1-42-induced oxidative stress and apoptosis in mouse hippocampal neurons.

Alzheimer's disease (AD) is a serious neurodegenerative disease. Senile plaques (SPs) composed of amyloid-ß (Aß) are typical features of AD. Aß plays a key role in the disease and has the ability to induce other pathological characteristics of AD, including oxidative stress injury. (m)VD-hemopressin (VD), a peptide derived from mouse brain extracts, can bind cannabinoid 1 receptor (CB1R) as an agonist. Our previous report indicated that VD reverses memory impairment induced by Aß1-42 in mice. This study aimed to clarify the mechanism by which VD protects hippocampal neurons against Aß1-42-induced impairment. Our results showed that VD inhibited oxidative stress injury induced by Aß1-42, as demonstrated by the VD-induced reversal of the upregulation of reactive oxygen species (ROS) and the intracellular lipid peroxidation product malondialdehyde (MDA) and the downregulation of the activities of the antioxidative enzymes catalase (CAT) and glutathione peroxidase (GSH-PX) in mouse hippocampal neurons. We also found that VD restored the decrease in cell growth and viability induced by Aß1-42 and reversed Aß1-42-induced apoptosis mediated by the apoptosis-associated proteins Bcl-2 and Bax. However, cotreatment with AM251 (an antagonist of CB1R) blocked the effects of VD. In brief, this study suggested that through CB1R, VD reversed the impairment of cell growth and viability, oxidative stress injury and apoptosis induced by Aß1-42. Therefore, VD may be a promising agent for the treatment of diseases that involve oxidative stress injury and apoptosis induced by Aß1-42, such as AD.

Stability and inhibitory function of Treg cells under inflammatory conditions in vitro.

Immunotherapy with transplanted T-regulatory (Treg) cells is currently in use. However, patients have complex internal environments with confounding factors, including the presence of inflammatory cytokines. The present study aimed to detect Treg cell function under simulated inflammatory conditions to provide a foundation for Treg cell-based immunotherapy. CD4+CD25high Treg cells were sorted from peripheral blood mononuclear cells and cultured for 14 days in the presence of recombinant human interleukin-2 (rhIL-2) and anti-CD3/CD28 beads, with or without 25 ng/ml rhIL-6. Next, the absolute count of Treg cells was determined, the stability and activity were detected by measuring the expression levels of forkhead box (Fox)P3 and CD39, and the suppressive function of Treg cells was investigated by assessing the suppression of T-effector cell proliferation by Treg cells after co-culture for 5 days. The number of Treg cells cultured in the presence of 25 ng/ml rhIL-6 for 14 days was reduced by 49.7% when compared with that of cells cultured without rhIL-6. Of the Treg cells continually cultured for 14 days without or with 25 ng/ml rhIL-6, 56.15 and 24.7% expressed FoxP3, respectively. There was no difference in the activity of the FoxP3+ Treg cells after culture for 14 days without or with 25 ng/ml rhIL-6. The suppressive function of Treg cells tended to deteriorate in the presence of rhIL-6. In conclusion, IL-6 inhibited the proliferation and stability of Treg cells, suggesting that administration of increased numbers of Treg cells may be required during Treg cell-based immunotherapy.

Design, synthesis, and biological evaluation of novel androst-17ß-amide structurally related compounds as dual 5α-reductase inhibitors and androgen receptor antagonists.

Prostate cancer (PCa) is the second leading cause of death in men. Apart from androgen receptor, 5α-reductase has also been recognized as a potential target. In this study, a series of androst-17ß-amide compounds have been designed and synthesized targeting both AR and 5α-reductase. Their anti-proliferation activities were evaluated in AR + cell line 22RV1 and AR - cell line PC-3. The results indicated that most of the synthesized compounds inhibited the testosterone-stimulated cell proliferation with good selectivity and safety. Among all the compounds, androst[3,2-c]pyrazole derivatives (9a-9d) displayed the best inhibition activity comparable with flutamide. Moreover, most of the synthesized compounds displayed good 5α-reductase inhibitory activities with IC50 lower than 1 µM. The docking result of 9d-AR indicated that AR was forced to expands its binding cavity and maintain an antagonistic conformation since the steric hindrance of 9d impeded H12 transposition. Overall, compound 9d can be identified as a potential dual 5α-reductase inhibitor and AR antagonist, which might be of therapeutic importance for PCa treatment.

Design and synthesis of novel steroidal imidazoles as dual inhibitors of AR/CYP17 for the treatment of prostate cancer.

Both AR and CYP17 are important targets for blocking androgen signaling, and it has been accepted that multifunctional drugs have a low risk of drug resistance in the treatment of cancer. Thus, herein a series of steroidal imidazoles were designed, synthesized and evaluated as dual AR/CYP17 ligands. Several compounds displayed good biological profiles in both enzymatic and cellular assays. SAR studies showed that introducing oximino at the C-3 position of steroidal scaffold is beneficial to the enhancement of AR antagonistic activity. Among these compounds, the most potent compound 13a exhibited the best AR inhibition (IC50 = 0.5 µM) that was 27-fold increase compared with the hit compound 5 as well as comparable CYP17 inhibition (IC50 = 11 µM). Additionally, 13a displayed promising anti-proliferative effects on LNCap cell lines with the IC50 value of 23 µM which was superior to positive control Flutamide (IC50 = 28 µM). Furthermore, the docking results of 13a revealed that the oxygen atom at the position of C-3 connected to the heme of CYP17, which may be helpful for its satisfactory dual-target inhibition. In summary, this study provides an efficient strategy for multi-targeting drug discovery in the treatment of prostate cancer.

Potential Astrocytic Receptors and Transporters in the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia and is characterized by the progressive loss of memory and cognition in the aging population. However, the etiology of and therapies for AD remain far from understood. Astrocytes, the most abundant neuroglia in the brain, have recently aroused substantial concern due to their involvement in synaptotoxicity, amyloidosis, neuroinflammation, and oxidative stress. In this review, we summarize the candidate molecules of astrocytes, especially receptors and transporters, that may be involved in AD pathogenesis. These molecules include excitatory amino acid transporters (EAATs), metabotropic glutamate receptor 5 (mGluR5), the adenosine 2A receptor (A2AR), the α7-nicotinic acetylcholine receptor (α7-nAChR), the calcium-sensing receptor (CaSR), S100ß, and cannabinoid receptors. We describe the characteristics of these molecules and the neurological and pharmacological underpinnings of these molecules in AD. Among these molecules, EAATs, A2AR, and mGluR5 are strongly related to glutamate-mediated synaptotoxicity and are involved in glutamate transmission or the clearance of extrasynaptic glutamate in the AD brain. The α7-nAChR, CaSR, and mGluR5 are receptors of Aß and can induce a plethora of toxic effects, such as the production of excess Aß, synaptotoxicity, and NO production triggered by changes in intracellular calcium signaling. Antagonists or positive allosteric modulators of these receptors can repair cognitive ability and modify neurobiological changes. Moreover, blocking S100ß or activating cannabinoid receptors reduces neuroinflammation, oxidative stress, and reactive astrogliosis. Thus, targeting these molecules might provide alternative approaches for treating AD.

3, 9-di-O-substituted coumestrols incorporating basic amine side chains act as novel apoptosis inducers with improved pharmacological selectivity.

There is much interest in the use of phytoestrogens such as coumestrol in breast cancer intervention due to their antiestrogenic activity and multiple modes of tumor cell death. However, the clear beneficial effects of naturally occurring estrogen mimetic coumestrol remain controversial due to experimental evidence that it has been shown to stimulate MCF-7 cell proliferation via agonist effect on estrogen receptor at low concentration. Herein, to disconnect the ER interaction and apoptosis-specific mechanism of coumestrol, various 3, 9-di-O-substituted coumestrols (7a-7e) and their furan ring-opened analogs (5a-5e) were synthesized and assessed for antiproliferative properties. Attachment of a dimethylamine-containing side chain to 3-O of coumestrol led to the most promising compound 7e with improved antiproliferative activity (1.7-fold increase) against MCF-7 cells, decreased estrogen activity (>20 times weaker ERα binder) and a novel action to induce apoptosis. Mechanistic studies revealed that 7e is a tubulin polymerization inhibitor, which could arrest cell cycle at G2/M phase and induce apoptosis along with the decrease of mitochondrial membrane potential. In summary, such subtle modifications to the 3, 9-di-hydroxyl groups of coumestrol allow the generation of a novel apoptosis inducer with distinct pharmacological properties, providing an excellent starting point to future development of novel tumor-vascular disrupting agents targeting tubulin.

Drug development for Alzheimer's disease: review.

Alzheimer's disease (AD) is a chronic neurodegenerative disease, which is considered as one of the most intractable medical problems with heavy social and economic costs. The current drugs for AD, including acetylcholinesterase inhibitors (AChEIs) and memantine, a NMDA receptor antagonist, only temporarily ameliorate cognitive decline, but are unable to stop or reverse the progression of dementia. This paper reviewed the recent advance in AD drug development. The drug discovery programs under clinical trials targeting cholinergic system, α7 nicotinic acetylcholine receptors (nAChRs), N-methyl-d-aspartate receptor (NMDAR), ß-secretase, γ-secretase modulators, tau, inflammatory mediators and glucagon-like peptide-1 (GLP-1) were discussed. Though several drug discovery programs are ongoing, the high failure rate is an outstanding issue. Novel techniques and strategies are desperately needed to significantly accelerate this process.

Aerobic exercise promotes the expression of ERCC1 to prolong lifespan: A new possible mechanism.

Aerobic exercise can delay aging and extend lifespan, but its specific mechanism still remains unclear. One popular theory is that with age and the cell division times increasing, DNA damage will inevitably accumulate, leading to dysfunction and failure of various tissues and organs, which will eventually lead to aging. Thus, repairing damaged DNA is a key strategy to extend lifespan. Excision repair cross-complementary gene 1 (ERCC1) is a DNA repair enzyme that recognizes, excises and repairs damaged DNA. Defects or reduced activity of the enzyme can lead to DNA damage accumulation. This study provides that aerobic exercise can significantly extend rats' lifespan and increase the expression of ERCC1 in heart, brain, liver and kidney. Therefore, based on our experiments, we propose the following scientific hypothesis: aerobic exercise can up-regulate the expression of ERCC1 and then may reduce DNA damage accumulation to maintain genomic integrity and stability, thereby delaying aging and prolonging lifespan in humans.

Aerobic exercise-stimulated Klotho upregulation extends life span by attenuating the excess production of reactive oxygen species in the brain and kidney.

Aerobic exercise induces many adaptive changes in the whole body and improves metabolic characteristics. Klotho, an anti-aging gene, is mainly expressed in the brain and kidney. The roles of Klotho in the brain and kidney during aerobic exercise remain largely unknown. The present study aimed to determine whether aerobic exercise could influence the expression of Klotho, decrease reactive oxygen species (ROS) and prolong life span. Sprague Dawley rats were exercised on a motor treadmill. Klotho mRNA and protein expression levels in rat brain and kidney tissues were examined using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. ROS production was detected following intermittent aerobic exercise (IAE) or continuous aerobic exercise (CAE). Kaplan-Meier curve analysis demonstrated that aerobic exercise significantly improved rat survival (P<0.001). The ROS levels in rat brain and kidney tissues were decreased in the aerobic exercise groups compared with the control group (P<0.05). In addition, Klotho mRNA and protein expression levels were increased significantly following aerobic exercise compared with controls (P<0.05). There was no significant difference between the IAE and CAE groups in any experiments (P>0.05). These results suggest that aerobic exercise-stimulated Klotho upregulation extends the life span by attenuating the excess production of ROS in the brain and kidney. As Klotho exhibits a potential anti-aging effect, promoting Klotho expression through aerobic exercise may be a novel approach for the prevention and treatment of aging and aging-related diseases.

Gpn2 and Rba50 Directly Participate in the Assembly of the Rpb3 Subcomplex in the Biogenesis of RNA Polymerase II.

RNA polymerase II (RNAPII) is one of the central enzymes in cell growth and organizational development. It is a large macromolecular complex consisting of 12 subunits. Relative to the clear definition of RNAPII structure and biological function, the molecular mechanism of how RNAPII is assembled is poorly understood, and thus the key assembly factors acting for the assembly of RNAPII remain elusive. In this study, we identified two factors, Gpn2 and Rba50, that directly participate in the assembly of RNAPII. Gpn2 and Rba50 were demonstrated to interact with Rpb12 and Rpb3, respectively. An interaction between Gpn2 and Rba50 was also demonstrated. When Gpn2 and Rba50 are functionally defective, the assembly of the Rpb3 subcomplex is disrupted, leading to defects in the assembly of RNAPII. Based on these results, we conclude that Gpn2 and Rba50 directly participate in the assembly of the Rpb3 subcomplex and subsequently the biogenesis of RNAPII.

Structure-activity relationships of 2, 4-disubstituted pyrimidines as dual ERα/VEGFR-2 ligands with anti-breast cancer activity.

Both ERα and VEGFR-2 are important targets for cancer therapies. Here a series of 2, 4-disubstituted pyrimidine derivatives were designed, synthesized and evaluated as dual ERα/VEGFR-2 ligands. Most of the derivatives exhibited potent activities in both enzymatic and cellular assays. Structure-activity relationship studies showed that a hydrogen-bonding interaction in the head section is important factors for the enhancement of ERα-binding affinity. The most potent compound II-9OH, an analog of 2-(4-hydroxylphenyl)pyrimidine, was 19-fold more efficacious than tamoxifen in MCF-7 cancer cells and exhibited the best ERα binding affinity (IC50 = 1.64 µM) as well as excellent VEGFR-2 inhibition (IC50 = 0.085 µM). Furthermore, this dual targeted compound II-9OH exerted significantly antiestrogenic property via suppressing the expression of progesterone receptor (PgR) mRNA in MCF-7 cells and also showed obvious in vivo angiogenesis inhibitory effects in CAM assay. An induction of apoptosis and a decrease in cell migration, accompanied by transduction inhibition of Raf-1/MAPK/ERK pathway, were observed in MCF-7 cells after treatment with II-9OH, suggesting that II-9OH is a promising candidate for the development of multifunctional agents targeting ERα and VEGFR-2 in the therapy of some breast cancers.

Design, synthesis and biological evaluation of novel 3-oxo-4-oxa-5α-androst-17ß-amide derivatives as dual 5α-reductase inhibitors and androgen receptor antagonists.

Prostate cancer (PCa) is the second leading cause of death in men. Recently, some researches have showed that 5α-reductase inhibitors were beneficial in PCa treatment as well. In this study, a series of novel 3-oxo-4-oxa-5α-androst-17ß-amide derivatives have been designed and synthesized in a more simple and convenient method. Most of the synthesized compounds displayed good 5α-reductase inhibitory activities and androgen receptor binding affinities. Their anti-proliferation activities in PC-3 and LNCaP cell lines were also evaluated and the results indicated that most of the synthesized compounds exhibited potent anti-proliferative activities. It is obvious that the androgen-dependent cell line LNCaP was much more sensitive than the androgen-independent cell line PC-3. Among all the synthesized compounds, 11d and 11k displayed the best inhibition activity with 4-fold more sensitive toward LNCaP than PC-3, which was consistent with their high affinities observed in AR binding assay. Molecular modeling studies suggested that 11k could bind to AR in a manner similar to the binding of dihydrotestosterone to AR. Compared to the finasteride, 11k showed a longer plasma half-life (4h) and a better bioavailability. Overall, based on biological activities data, compound 11d and 11k can be identified as potential dual 5α-reductase inhibitors and AR antagonists which might be of therapeutic importance for prostate cancer treatment.

Design, synthesis and biological evaluation of novel 2-methoxyestradiol analogs as dual selective estrogen receptor modulators (SERMs) and antiangiogenic agents.

2-methoxyestradiol is a novel agent showing both anti-angiogenic and vascular disrupting properties. In this study, a series of 11α-substituted 2-methoxyestradiol analogs have been designed and synthesized targeting dual ERα and microtubulin. Biological evaluation was performed on their anti-proliferative activities against 5 different cell lines. The results indicated that most compounds exhibited good activities, in which compound 24c and 30c showed the best activity with low micromolar IC50 (2.73 µM -7.75 µM) in all cell lines. The investigation of ER affinity showed that the majority of the compounds displayed good activity at the concentration of 50 µM. In further mechanism study, it was observed that 24c and 30c could induce G2/M cell cycle arrest as well as significant anti-estrogenic activity. In CAM assay, compound 24c and 30c presented significantly anti-angiogenesis activity comparable with 2-methoxyestradiol. Overall, based on biological activities data, 24c and 30c can be identified as a potential lead molecule which might be of therapeutic importance for cancer treatment.

Design, synthesis and biological evaluation of novel androst-3,5-diene-3-carboxylic acid derivatives as inhibitors of 5α-reductase type 1 and 2.

5α-Reductase is a key enzyme responsible for dihydrotestosterone biosynthesis and has been recognized as an important target for discovering new drugs against benign prostatic hyperplasia (BPH). In this study, a series of novel steroidal androst-3,5-diene-3-carboxylic acids have been designed and synthesized. Biological evaluations were performed on their 5α-reductase inhibitory activities by both in vitro enzyme inhibition assay and in vivo by prostate weighing method. Results showed that most of them displayed excellent 5α-reductase inhibitory potency. Detailed evaluation indicated that most of the compounds displayed slightly higher inhibition potency towards type 2 isozyme. Among all the compounds, 16a was found to be the most potential inhibitor with the IC50 of 0.25µM and 0.13µM against type 1 and 2 isozymes respectively. In vivo 5a-reductase inhibitory evaluation of 16a also showed a more significant reduction effect (p<0.001) in rat prostate weight than epristeride. Furthermore, the results of in silico ADME study indicated that compound 16a exhibited good pharmacokinetic properties. Thus, 16a could serve as promising lead candidates for further study.